Structure of Bovine Rhodopsin in a Trigonal Crystal Form

doi:10.1016/j.jmb.2004.08.090 J. Mol. Biol. (2004) xx, 1–30

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Structure of Bovine Rhodopsin in a Trigonal CrystalForm

Jade Li1*, Patricia C. Edwards1, Manfred Burghammer2, Claudio Villa1

and Gebhard F. X. Schertler1*
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1Medical Research CouncilLaboratory of Molecular BiologyHills Road, Cambridge CB22QH, UK

2European SynchrotronRadiation Facility, BP 2208043 Grenoble, France

UNCO

0022-2836/$ - see front matter q 2004 P

Abbreviations used: 2D, two-dimloop connecting helices 1 and 2; C2,5 and 6; cGMP, 3 0,5 0-cyclic guanosinconnecting helices 2 and 3; E2, extraand 7; EM, electron microscopy; EMGPCR, G protein-coupled receptor;transducin a-subunit; Gta 340–350,N-oxide; NCS, non-crystallographicE-mail addresses of the correspon

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ECTED PROOF

We have determined the structure of bovine rhodopsin at 2.65 A resolutionusing untwinned native crystals in the space group P31, by molecularreplacement from the 2.8 A model (1F88) solved in space group P41. Thenew structure reveals mechanistically important details unresolvedpreviously, which are considered in the membrane context by dockingthe structure into a cryo-EM map of 2D crystals.

Kinks in the transmembrane helices facilitate inter-helical polarinteractions. Ordered water molecules extend the hydrogen bondingnetworks, linking Trp265 in the retinal binding pocket to the NPxxY motifnear the cytoplasmic boundary, and the Glu113 counterion for theprotonated Schiff base to the extracellular surface. Glu113 forms a complexwith a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt bridge withthe protonated Schiff base linking the 11-cis-retinal to Lys296.

The cytoplasmic ends of helices H5 and H6 have been extended by oneturn. The G-protein interaction sites mapped to the cytoplasmic ends of H5and H6 and a spiral extension of H5 are elevated above the bilayer. There isa surface cavity next to the conserved Glu134-Arg135 ion pair. Thecytoplasmic loops have the highest temperature factors in the structureindicative of their flexibility when not interacting with G-protein orregulatory proteins. An ordered detergent molecule is seen wrappedaround the kink in H6, stabilising the structure around the potential hingein H6.

These findings provide further explanation for the stability of the darkstate structure. They support a mechanism for the activation, initiated byphoto-isomerisation of the chromophore to its all-trans form, that involvespivoting movements of kinked helices, which, while maintaining hydro-phobic contacts in the membrane interior, can be coupled to amplifiedtranslation of the helix ends near the membrane surfaces.

q 2004 Published by Elsevier Ltd.

Keywords: G protein-coupled receptor; G protein activation; ligand bindingpocket; membrane protein structure; visual pigments

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R*Corresponding authors
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ublished by Elsevier Ltd.

ensional; 3D, three-dimensional; C8E4, n-octyltetraoxyethylene; C1, cytoplasmiccytoplasmic loop connecting helices 3 and 4; C3, cytoplasmic loop connecting helicese monophosphate; cryo-EM, electron cryomicroscopy; E1, extracellular loopcellular loop connecting helices 4 and 5; E3, extracellular loop connecting helices 6TS, ethyl mercurythiosalicylate; FTIR, Fourier transform infrared spectroscopy;G-protein, heterotrimeric guanine nucleotide binding protein; Gt, transducin; Gta,C-terminal peptide from transducin a-subunit; LDAO,N,N-dimethyldodecylamine-symmetry; PDE, phosphodiesterase.ding authors: [email protected]; [email protected]

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2 Rhodopsin Structure in Trigonal Crystals


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UNCORREC

Introduction

G protein-coupled receptors (GPCRs) constitutethe largest superfamily of transmembrane signal-ling proteins in the eukaryotic kingdom. They sharea common core structure comprising seven trans-membrane helices, as implied by a conservedpattern of amino acids at key positions along eachof the seven hydrophobic sequence segments.1

These proteins capture external signals includinglight, odorants, hormones and neurotransmitters,and transmit the stimuli across the plasmamembrane by selectively activating heterotrimericguanine nucleotide binding proteins (G proteins) onthe cytoplasmic surface. Activation of G proteinamplifies the signal and leads to activation ofeffector enzymes or molecules, which elicit thecell’s signalling response indirectly by alteringsecond messenger concentrations or by directcontrol of ion channel activities. Thus, the GPCRsplay a central role in regulating many physiologicalprocesses. They are consequently major targets fordrug design.

Rhodopsin, the photoreceptor protein in retinarod cells, is a prototypical GPCR. It contains a light-sensitive ligand, the 11-cis-retinal chromophore,bound covalently to the apoprotein opsin via aprotonated Schiff base with Lys296 on helix 7.Absorption of a photon at about 500 nm isomerisesthe retinal to all-trans2 within picoseconds and witha quantum efficiency of 0.67. This event initiates theformation of a series of photointermediates3 withconformational changes in the opsin.4 The bio-chemically active conformation R* is attainedwithinone millisecond at physiological temperature. It isspectroscopically identified with the metarhodop-sin II (MII) intermediate, which has an absorptionmaximum at 380 nm due to deprotonation of theSchiff base of all-trans-retinal. R* binds and activatesthe heterotrimeric G-protein transducin (Gt) at thecytoplasmic surface, to catalyse GDP/GTPexchange on the Gt a-subunit and dissociation ofthe Gt heterotrimer. The GTP-bound a-subunit thenactivates the effector enzyme, phosphodiesterase(PDE). Hydrolysis of cyclic guanine monophos-phate (cGMP), the second messenger, by PDE leadsto closure of the cGMP-gated cation channel in theplasma membrane, causing hyperpolarisation andinitiation of nerve impulse in the retina.5 In contrastto the efficient and rapid photoisomerisation andactivation, the rate of thermal isomerisation is verylow, about one per 400 years per rhodopsinmolecule at 37 8C.6 The low thermal noise coupledwith large amplification via transducin activationunderpins the sensitivity of single photon detectionoperating in dim light vision.

Anatomic structure of rhodopsinprovides amodelfor all the visual pigments as well as the majority ofthe GPCRs. Crystallisation in two-dimensional (2D)lattices with endogenous membrane lipids, followedby electron cryo-microscopy (cryo-EM)7–11 atresolution limits up to 5 A in the membrane planeand 13.5 A normal to it, have produced images of

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D PROOF

bovine, frog and squid rhodopsins in a membrane-like environment, which are in essence similar. Usingpacking constraints derived by extensive sequencecomparisons across the GPCR superfamily, thedensity peaks in cryo-EM maps were assigned tohydrophobic sequences, leading eventually to aC-alpha model for the seven transmembrane helicesin the rhodopsin-likeGPCR family.1,12,13 Remarkably,this model came within 2.3 A rms deviation of Ca

coordinates determined by X-ray crystallographysubsequently14,15 and it provided a framework formutagenesis andbiophysical studies in theabsenceofan atomic structure.

To determine the atomic structure of rhodopsin,we have obtained untwinned three-dimensionalcrystals of bovine rhodopsin in the trigonal spacegroup P31 that diffracted X-rays to 2.65 Aresolution, and prepared an ethylmercury deriva-tive that showed anomalous scattering.16 However,significant non-isomorphism prevented experi-mental phasing. While this work was in progress,a structure of bovine rhodopsin in a tetragonal P41space group at 2.8 A resolution was reported.14,17

Using these coordinates as search model, we havedetermined the structure of bovine rhodopsin to2.65 A resolution in the P31 lattice by molecularreplacement followed by multiple crystal averagingamong the non-isomorphous data sets. Here wedescribe the refined structure of bovine rhodopsinin the trigonal crystal form, and compare it withprevious structural reports of the tetragonal crystalform.14,18,19 In addition, we relate the crystalstructures to the membrane environment bydocking them into a cryo-EM map10 of 2D crystals.Structural implications for the stability of the“dark” state and the potential for light-inducedconformational change, leading to transducinactivation on the cytoplasmic surface, arediscussed.

Results and Discussion

Structure determination

Bovine rhodopsin was crystallised in the trigonalcrystal space group P31 with two protein moleculesper asymmetric unit, from a detergent mixture ofC8E4 and LDAO as described in the accompanyingpaper.16 Data sets were obtained from native andmercury-derivatised crystals prepared and frozenunder dim red light, however they showed signifi-cant non-isomorphism with one another. Datacollection and refinement statistics are given inTable 1.

Using the protein part only of the rhodopsincoordinates solved in a tetragonal P41 space group(PDB code 1F88)14,17 at 2.8 A resolution as searchmodel, molecular replacement solutions werecalculated in parallel for three sets of amplitudes(Native-2, EMTS-1 and EMTS-2). The resulting2FoKFc maps showed no density in three cyto-plasmic regions of 1F88 coordinates, and the

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CTED PROOF

Table 1. Data collection and final refinement statistics

(A) Data collection

Native-2a EMTS-1 EMTS-2 Native-3

X-ray source ESRF ID13 ESRF ID13 ESRF ID14-4 ESRF ID13Wavelength (A) 0.782 1.005 1.0065 0.782Unit cell a, c (A) 104.2, 77.1 113.9, 78.4 109.3, 77.6 103.8, 76.6No. of crystals 2 1 1 4Mosaicity (deg.) 1.0 1.1 1.1 0.75Twin fraction 0 0 0.31 0Resolution (A) 3.2 (3.37–3.20) 3.6 (3.71–3.60) 3.4 (3.45–3.40) 2.65 (2.79–2.65)Rmerge

b 0.127 (0.322) 0.169 (0.464) 0.139 (0.426) 0.119 (0.434)I/s 7.5 (2.3) 7.6 (2.2) 17.2 (4.7) 11.0 (1.4)Unique reflections 15087 11481 13320 26026Completeness (%) 0.982 (0.894) 0.868 (0.180) 0.834 (0.172) 0.970 (0.861)Multiplicity 3.1 (1.6) 5.3 (2.9) 11.3 (9.5) 4.4 (1.6)Wilson B (A2) 84.6 59.7 54.2 58.2

(B) Refinement against Native-3

Reflections in working set 24704 (2165) Protein chains 2Reflections in test set 1322 (130) Protein residues 652Resolution range (A) 46–2.65 (2.74–2.65) Palmitoyl chains 4Rcryst

c 0.202 (0.312) N-linked carbohydrate chains 4Rfree

d 0.235 (0.315) Carbohydrate residues 12Luzzati coordinate error (5.0–2.65 A) 0.31 A LDAO 2SigmaA coordinate error (5.0–2.65 A) 0.42 A C8E4 12Rms-deviation from ideal geometry Phospholipid 2Bond lengths (A) 0.008 Water 40Bond angles (deg.) 1.293 Ions 2Dihedral angles (deg.) 18.7Improper rotations (deg.) 0.876Ramachandran plot% Residues in most favoured regions 90.6% Residues in additional allowed regions 7.1% Residues in generously allowed regions 2.4% Residues in disallowed regions 0Average B-factor (A2) 56.0B rmsd for bonded main chain atoms (A2) 1.501B rmsd for bonded side chain atoms (A2) 1.996B rmsd for angle main chain atoms (A2) 2.624B rmsd for angle side chain atoms (A2) 3.134

Values in parenthesis apply to the outer resolution shell.a Names of data sets conform to Table 1 of the accompanying paper.149 Native-1 is not referred to here, because it was not used in the

structure determination.b RmergeZ

Phkl

Pi jIiK hIij=

Phkl

Pi Ii, where Iiis the intensity of an individual reflection and!IO is themean intensity obtained from

multiple observations of symmetry related reflections taken from one or more crystals.c P

hklðjFoðhklÞKFcðhklÞjÞ=P

hklðjFoðhklÞjÞ, calculated for reflections in the working set.d P

hklðjFoðhklÞKFcðhklÞjÞ=P

hklðjFoðhklÞjÞ, calculated for a random 5% of the data placed in the test set.

Rhodopsin Structure in Trigonal Crystals 3


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UNCORREcorresponding FoKFc maps showed negative peaksin the same regions. These regions were: the C2 loopconnecting transmembrane helices H3 and H4, theC3 loop connecting H5 and H6, and the C-terminaltail of residues 334–348. By contrast, density waspresent in the 2FoKFc maps over positions of theretinal chromophore, even though, as a control, wehad omitted the retinal from the 1F88 coordinatesused for molecular replacement. No negative peakswere seen in the retinal-binding pocket. Thereforeour amplitude data disagreed with the startingmodel in the cytoplasmic regions. Exploiting thelarge non-isomorphism, we carried out cross-crystal averaging from these data sets. Theaveraged map for Native-2 showed densities forthe C2 and C3 loops at positions different from thestarting model, enabling them to be rebuilt and thecytoplasmic end of helix H6 to be extended from

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residue 247 in the 1F88 coordinates to residue 244.The middle section of the C3 loop (residues 233–239in molecule A and 233–237 in molecule B) and the Cterminus still had no interpretable densities, so theywere left out of the model. The Schiff base linked11-cis-retinal chromophore was built from the smallmolecule crystal structures20,21 by adjusting torsionangles along the polyene according to the averageddensity.A new native dataset (Native-3) was then

collected from four untwinned crystals to 2.65 Aresolution, and used to refine the atomic model,using CNS22 under 2-fold non-crystallographicsymmetry restraints with simulated annealing andrestrained individual B-factors. Bound watermolecules were located using the water-pick taskof CNS22 and confirmed in maps calculated usingEDEN.23 Tightly bound phospholipids and

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detergent molecules were added. Including thebound water and amphiphile molecules in therefinement further improved the map, so that thecytoplasmic ends of both H5 and H6 were extendedand the C3 loop was completed. The most signifi-cant differences between our structure and all thepublished coordinates from the P41 crystal arefound in this region.

Quality of the structure

The final crystallographic R-factor is 0.202 with afree R-factor of 0.235, and there is close agreementwith ideal geometry (Table 1B). The Ramachandranplot contains 90% of the protein residues in themost favoured regions, compared with values of80.9–82.5% for the published rhodopsin structuresin the tetragonal crystal form. The SIGMAA-weighted24 2FoKFc map showed an average real-space correlation of 0.95 for all residues includingthe non-protein residues. These statistics demon-strate that the structure was well determined, andbias toward the starting model was minimal.

The refined model (Table 1B) comprises aminoacid residues 1–326 in both molecules, comparedwith the complete sequence of 348 residues.Post-translational modifications have been definedon both molecules, includingN-acetylation of Met1,N-glycosylation of Asn2 and Asn15, palmitoylationof Cys322 and Cys323, and the Schiff base linkage of11-cis-retinal to Lys296. In addition, there are 20ordered water molecules bound to each proteinchain and a number of tightly bound lipid anddetergent molecules. The covalent structure isshown as a ribbon diagram in Figure 1(a). Theresidue ranges of the seven transmembrane helicesare: H1, 34–64; H2, 71–100; H3, 106–140; H4,150–173; H5, 200–230; H6, 241–276 and H7,286–309. Residues in the peripheral helix H8 are311–321, and those making up the b-strands are: b1,4–6; b2, 9–11; b3, 177–180 and b4, 187–190. Portionsof the transmembrane helices, H4 between residues170 and 173 and H7 between residues 296 and 299,are in the 310 conformation.

Comparison with the tetragonal crystal form
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UNCORRPacking differences

Both the P31 and P41 crystal forms contain tworhodopsin molecules per asymmetric unit. In theP41 form, they are related by a non-crystallographicrotation of 172.58 about an axis nearest to a and areoriented obliquely from the P41 axis. They makecontacts in the middle of helix 1 but mainly overthe hydrophilic extra-membrane segments. Inparticular, the C terminus of molecule A from thecytoplasmic domain forms contact with theextracellular domain of molecule A in anotherasymmetric unit.

In the P31 form, the non-crystallographicsymmetry (NCS) is a nearly perfect 2-fold aboutan axis parallel to the 31 axis and passing through


F

(xZ0.333, yZ0.167). Both helix bundles are tilted by1088 from c and make antiparallel contacts along theentire length of helix 5. The antiparallel dimers arestacked according to the 31 screw symmetry,forming two protein columns in the unit cell,which are centred on two of the three 3-foldpositions of the trigonal lattice, and leaving asolvent channel centred on the remaining 3-foldposition. Between layers of stacked dimers thecontacts involve mainly the sides of helices 1 and 4.The other helices participate in crystal contacts to alesser extent or indirectly via interleaved palmitoy-late chains or the C8E4 molecule (see subsectiontitled Bound lipid and detergent molecules). Thus,uncommonly among crystals of membrane pro-teins, the inter-molecular contacts in the P31 formare predominantly hydrophobic. The hydrophilicextra-membrane segments point into the solventchannel and are not responsible for ordered crystalcontacts (Figure 1(b)). Consequently, the P31 crystalpacking has retained an amphipathic molecularenvironment characteristic of the native membrane,permitting the hydrophilic segments to displayconformations present in a membrane environmentunhindered by packing interactions.

ED PROO

Coordinate differences between NCS copies andbetween crystal forms

The 1L9H coordinates19 at 2.6 A resolution areused to represent the P41 form. Here, the rms Ca

distance between NCS copies is 0.470 A for 301matched residues, and the rms B-factor difference is9.530 A2 with molecule A being the more orderedcopy. In the P31 form with closer NCS, the twomolecules show differences of only 0.13 A and2.216 A2 for 326 matched residues. Thereforemolecule A from the 1L9H coordinates and thepresent structure are used to compare the crystalforms.

Figure 2(a) shows the coordinate difference perresidue as a function of sequence position, betweenNCS copies in each crystal form and between thetwomolecules A of crystal forms. Both crystal formsshowed significant main chain NCS differences,above 1 A, in the C2 and C3 loops and theC-terminal tail following helix 8. These regionsalso have the highest B-factors and disorder in eachcrystal form (Figure 2(b)). Part of the C3 loop isunresolved in both molecules of the P41 form. TheC-terminal tail in the P31 form is located in thesolvent channel and disordered, while in the P41form it is partially resolved only in molecule A,where it is stabilised through contacts with theextracellular domain of a neighbouring molecule.Finding significant NCS differences in the samesurface segments indicates an intrinsic confor-mational variability or flexibility of those segments,and the high B-factors of the segments support this.These segments are known to contain residues thatinteract with transducin (Gt) and the regulatoryproteins rhodopsin kinase and arrestin;25–30 their

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RECTED PROOF

Figure 1. The bovine rhodopsin molecule in the transmembrane orientation with cytoplasmic surface at the top andextracellular surface at the bottom. (a) Ribbon representation. The seven transmembrane and one peripheral helices arecoloured in the rainbow order: H1 (residues 34–64), dark blue; H2 (71–100), light blue; H3 (106–140), blue-green; H4(150–173), yellow-green; H5 (200–230), yellow; H6 (241–276), orange; H7 (286–309), red; H8 (311–321), magenta. The b-strands are coloured in cyan. They are arranged in the order of b1 (4–6), b2 (9–11), b3 (177–180), and b4 (187–190), fromthe extracellular surface into the retinal-binding pocket. The transmembrane helices are kinked around the 11-cis-retinalto form the binding pocket. Also shown are the N-linked oligosaccharides attached to Asn2 (near b1) and Asn15, and thepalmitoylate chains attached to Cys322 and Cys323 after H8. This colour scheme for the secondary structures is usedthroughout. (b) Space filling representation. Residues coloured in yellow form the inter-molecular contacts in the P31crystal form, directly or via ordered lipid and detergent molecules. They are all located in the transmembrane helices.The oligosaccharides are coloured pink, and the Ca trace is shown in grey for comparison with (a). Figures 1, 2(b), 4, 5, 6,7(b) and 9(a) were prepared using Molscript;146 Figures 1, 2(b) and 9(a) also using Raster3D.147



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UNCORflexibility in the absence of the binding partnersmay have a functional role.

If main chain atoms in the molecules A of the twocrystal forms are superimposed by aligning themore ordered regions selected by a B-factor cut-offof 90 A2, then the rms coordinate difference is0.36 A, which is slightly less than the meancoordinate error for all atoms in either structure.Therefore the two structures are in good agreementin the ordered core of the molecule, whichcomprised residues 1–137, 154–223 and 250–321,and excluded only the neighbourhood of the C2 andC3 loops, and the C-terminal tail. The backbonedifferences are concentrated in the same regionsshown by the NCS comparisons to be intrinsically


more flexible. Figure 2(a) shows that, for the C2loop, the difference between crystal forms is on thesame scale as the NCS difference within the P41form, so they are accountable by local flexibility andpacking contacts. However, around the C3 loop thedifferences between the two crystal forms are muchlarger than the NCS differences in either form,therefore they indicate statistically significantdisagreement.

Location of major coordinate differences betweencrystal forms

Figure 2(b) shows the Ca trace of the molecules Afrom the two crystal forms laid side-by-side,

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Figure 2. (legend opposite)

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coloured by the mean B-factors averaged for eachresidue. The colour scheme shows that the newstructure from the P31 crystal form has the lowerB-factors everywhere except in the C3 loop, which isabsent in the other structure, and the majordifferences lie in the regions of higher B-factors.

The C2 loop has a similar “L-shape”31 in bothstructures but with a different orientation relative tothe helix bundle. The difference may be describedas a hinge movement about the junctions with thecytoplasmic ends of H3 and H4.

The C3 loop and the cytoplasmic ends of H5 andH6 show the most striking difference between thetwo structures. In molecule A of the P41 form, H5terminates at residue 226, following which thepolypeptide chain re-enters the bilayer and appearsdisordered between residues 236 and 240, while H6terminates at residue 244. In the P31 form, H5extends to residue 230, then the Ca trace continuesin a helix-like spiral path away from the membraneto residue 236, where it changes direction to runparallel to the membrane and joins the cytoplasmicend of H6 at residue 241. The cytoplasmic tip of H6is slightly bent at residue 244 towards H5.

The C-terminal tail between residues 334–348 inthe P41 structure adopts an extended conformationsupported by contacts with a neighbouringmolecule. In the P31 structure this segment isdisordered, except for a di-peptide suggested bythe density in contact with the cytoplasmic loop 1(loop C1). The disorder can be expected since thefragment, without disulphide bridges, is too smallto form an independently folded domain, and it islocated in a solvent channel in this crystal form.

The structure of the cytoplasmic domain of theP31 form is discussed at greater length in the contextof explaining the surface accessibility and mobilityobserved in site-specific spin labelling, chemicallabelling and cross-linking studies,32–35 as well asthe relationship to G-protein activation,36–39 in thesection of that name.
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ORRECOther differences between the crystal forms
The P31 structure shows mechanistically impor-tant differences from the 1L9H coordinates in theconformation of the covalently linked 11-cis-retinalchromophore (see subsection titled Conformationand environment of the chromophore). All theinternal water molecules have now been located,thus leaving no cavity18 in the protein. The eightnewly identified water molecules include one inthe vicinity of Trp265, and they augment the

UNCFigure 2. Comparison between the P31 and P41 crystal fo

position. The main chain and side-chain differences betwsuperimposed on the main chain difference between NCS coonly around the C2 loop (residues 141–149) connecting H3 andH6, and in the C-terminal tail following H8. (b) Ca trace of themean residue B-factors. The two structures differ in the oriconformation of the C3 loop and the C-terminal fragment.highest B-factors.


inter-helical H-bonding network (see subsectiontitled Transmembrane domain). The N-linkedcarbohydrate chains do not form ordered crystalcontacts in the P31 crystals, unlike in the P41 crystals(see section titled Extracellular domain).

ED PROOF

Placing the X-ray structure in the membranecontext

Before coordinates from the tetragonal 3Dcrystals became available, we were able to deter-mine the orientation of rhodopsin molecules in theP31 crystals by rotation search at 5 A resolution,16

using as search model the density for a singlemolecule masked from a cryo-EM map of 2Dcrystals10 (see Methods). Conversely, by dockingthe refined X-ray coordinates into this cryo-EMmap,10 we placed the atomic structure in themembrane context of the 2D crystal. The height ofrhodopsin relative to the bilayer was thereforeascertained, since the p22121 symmetry of those 2Dcrystals requires symmetrical insertion of moleculesfrom both faces of the bilayer and therefore theplane defined by cZ0 is equivalent to the midplaneof the bilayer.Figure 3(a) shows the mean residue height in the

bilayer as a function of sequence position, overlaidby the mean residue B-factors of the refined P31structure. The sequence ranges of the a and bsecondary structure elements are shown as verticalbars. Small-angle X-ray and neutron scattering haveshown that phosphate peaks in the retinal discmembranes are separated by 40 A across thebilayer,40,41 and lipid head groups in modelmembranes occupy a layer about a 10 A-thickcentred on the phosphate peaks.42 Therefore inFigure 3 the hydrophobic zone extends fromapproximately K15 to C15 A, and the lipid headgroup layers extend from there to approximatelyK25 and C25 A. On this scale, the 33 N-terminalresidues preceding H1 fall entirely outside thehydrophobic zone. They form the extracellulardomain. However only the residues 1–5 and13–21, which carry the N-linked carbohydratechains on Asn2 and Asn15, reach into the intra-discal space.On the extracellular face all transmembrane

helices terminate in the lipid head group layer,except H4, which terminates within the hydro-phobic zone. The strand b3 lies at the interfacebetween hydrophobic zone and head group layer,while b4 is contained within the hydrophobic zone.On the cytoplasmic face, helices H1, H2, H4 and H7

rms. (a) Coordinate difference as a function of sequenceeen the molecules A from different crystal forms arepies within each crystal form. Significant differences existH4 and the C3 loop (residues 231–240) connecting H5 andmolecules A from the two crystal forms coloured by theirentation of the C2 loop, the lengths of H5 and H6, theRegions of maximal structural differences also show the

30/TL

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UNCORRECTED PROOF

Figure 3. Relation of the P31 crystal structure to the membrane environment. (a) Mean residue height relative to thebilayer derived by docking the X-ray coordinates in the cryo-EM map of 2D crystals, with mean residue B-factorssuperimposed. Themid-plane of the bilayer is defined by the cZ0 plane of the p22121 2D lattice. The hydrophobic zone isestimated to lie in a 30 A band about this plane, and the lipid headgroups in two 10 A wide layers outside that. Thesecondary structure assignments are represented by vertical bars, with notches in the bars for H4 and H7 indicating thelocation of 310 segments. The curve of residue heights follows a straight path over sections of H4 and H6 that areperpendicular to the bilayer, but shows a zigzag pattern for the titled sections. Surperimposed on this curve is the meanB-factor per residue as a function of the sequence. (b) Mean residue B-factor as a function of transmembrane position.The B-factors are well clustered between the lipid phosphate positions at G20 A. They increase from the membraneinterior towards the surface, but the lowest B-factors are found just to the extracellular side of the bilayer mid-plane, atthe depth where the retinal is bound. The sharp rise of B-factors on the cytoplasmic surface is due to conformationalvariability in several regions.




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Table 2. Tilt and kinks in the transmembrane helices

Helix Straight segmentsa Residue rangeaTilt from membrane

normalbAzimuthal orientationc

(deg.)Kink angle

(deg.)

H1 N-segment 35–54 23 13 9C-segment 55–65 30 2

H2 N-segment 70–87 K24 K85C-segment 88–100 K33 K14 33

H3 N-segment 106–113 34 55Midsegment 113–127 32 77 12C-segment 127–139 24 80 8

H4 N-segment 150–169 K4 67C-segment 169–173 K33 1 35

H5 N-segment 200–211 20 8C-segment 211–231 26 36 13

H6 N-segment 244–265 K3 9C-segment 265–279 K34 K53 35

H7 N-segment 286–296 25 62Midsegment 297–302 4 13 27C-segment 303–309 18 4 14

H8 311–321 K86 89

a Residue range of the straight segments were determined by fitting cylinders of 2.8 A radius over the Ca positions. All segments havea-helical conformation except for the C-segment of helix 4 and residues 297–299 within the mid-segment of helix 7 which have the 310conformation.

b The membrane normal was taken from the c-axis of the 2D crystal in the two-sided plane group p22121. Positive tilt angle means thehelix runs from the extracellular side towards the cytoplasmic side as the sequence number increases.

c Angle from the a-axis of the p22121 lattice to the projection of helix segments in the plane of the 2D crystals, by a right-handedrotation about the membrane normal.



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terminate in the head group layer, whereas H3, H5and H6 reach into the cytoplasmic space. Residues66, 138–141, 144–147 and 229–248 from the threecytoplasmic loops are well exposed above the lipidhead group layer (Figure 3(a)) and available forinteractions with cytoplasmic proteins.30,38,39 Theperipheral helix H8 is partitioned between the headgroup layer and the cytoplasm and is slightlyinclined with its N terminus more exposed to thecytoplasm than its C terminus. The polypeptidefollowing H8 turns to the cytoplasm, but thepalmitoylate chains on Cys322 and Cys323 areinserted into the hydrophobic zone.

Transmembrane helices in rhodopsin are kinked(see subsection Transmembrane domain), and onlythe N-terminal segments of H4 and H6 are orientedroughly normal to the membrane (Table 2).Figure 3(b) shows that the mean residue B-factorsare strongly correlated with the depth in the bilayer,irrespective of the helix tilt angles. The lowestB-factors on average are found not at the midplaneof the bilayer midplane but in a 4 A band to theextracellular side of it. Therefore the dark staterhodopsin structure is most ordered at that depth inthe membrane, which coincides with where theionone ring of the 11-cis-retinal chromophoreis located. Beyond the phosphate head grouppositions (G20 A), the B-factors rise distinctlymore rapidly on the cytoplasmic surface than inthe extracellular domain, which is due to confor-mational variability in several regions.

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UExtracellular domain

The extracellular domain includes the short b1-b2hairpin, which together with the b3-b4 hairpin in


ED PROOthe transmembrane domain (Figure 1(a)) has been

described as forming a “plug” for the chromophore-binding pocket.14 The two b-hairpins are cross-linked by water-mediated H-bonds between mainchain atoms of Tyr10 in b2 and Pro180 in b3(Figure 4). Three water molecules (3, 5 and 13) areresponsible for this, of which one (13) was identifiedin the P41 structure.

19 These water molecules formadditional H-bonds to Gly182 and Gln184 in theb3-b4 hairpin and to Tyr192 near b4, further linkingthe b1-b2 hairpin to the transmembrane domainand securing the plug. Between Pro12 at the end ofb2 and Pro23, the polypeptide forms an outward“horn” (Figures 1(a) and 4) which is rigid with aturn (residues 15–18) at the apex and several side-chain -to main chain H-bonds. The horn wassuggested to act as a spacer between oppositefaces of the retinal disc,43 but the N-linkedoligosaccharides on Asn2 and Asn15 may alsocontribute in this role (Figure 1(a)). In our structurethere is density for only three residues in each of theoligosaccharide chains on Asn2 and Asn15:44

GlcNAc-(b1,4)-GlcNAc-(b1,4)-mannose. The distalcarbohydrate residues cannot be resolved, probablybecause they adopt heterogeneous orientationswithin the solvent channel. The orientations of theoligosaccharide chains relative to the protein differfrom those in the P41 structure, in which theywere found to form intermolecular contacts. Theremainder of this domain contains two turns(residues 22–25, 28–32) in the lipid head grouplayer adjacent to the transmembrane domain.Many H-bonds and van der Waals interactions

exist between the extracellular domain and the baseof the transmembrane domain, lending credence tothe view that the N-terminal domain may fold first

30/TL

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ROOF

Figure 4. Interface between the extracellular and transmembrane domains. Loops in the two domains are colouredblue and yellow, respectively. Note the water-mediated H-bonds linking b2 and b3, the stacking of Pro12 with Pro285and Pro27 with Tyr102, and the environment of Pro23. Amino acids are designated by one-letter codes for clarity inFigures 4–10.

† http://www.gpcr.org/7tm/



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UNCORRECand provide a template to assist the assembly of thehelical domain.45 Figure 4 shows that Asn2 is incontact with Gly280 and Asp282 in the E3 (H6-H7)loop, and Pro12 is stacked with Pro285 in the sameloop. Pro27 is stacked with Tyr102 in the E1 (H2-H3)loop, which is completely conserved amongvertebrate opsins. Pro23 and Gln28 form mainchain H-bonds with Gly101 and Tyr102. Pro23, themost conserved (99%) residue in the extracellulardomain of vertebrate opsins, is in a buried turn andin contact with Phe9 and Val11 in b2, and Gln28, aswell as Tyr102 and Phe103 in the E1 loop and Pro180and Gln184 in the E2 loop. Mutations of Pro23 canprevent correct folding of both domains. Pro23His,the most frequent mutation in American patientswith retinitis pigmentosa, has been shown to causeaggregation of misfolded rhodopsin, which istargeted for degradation by the ubiquitin proteo-some pathway in cultured cells but also impairs thedegradation pathway, and that impairment mayunderlie the neurodegenerative phenotype.46

Sequences of visual pigments are highly con-served and, relative to vertebrate opsins, the


Einvertebrate opsins show few insertions (GPCRDBdata base†). The longest insertion, of 13 residues, islocated in the turn of the b1-b2 hairpin; the others,of two, one and five residues each, are found afterresidue 332. Within the b1 and b2 strands visualpigments share around 30% sequence identity. Atthis level of similarity the invertebrate opsins can beexpected47 to contain a similar b-hairpin andaccommodate the insertion on the extracellularsurface (Figure 3(a)).

Transmembrane domain

Fitting cylinders of 2.8 A radius to the Ca trace inO48 divided the transmembrane helices into two orthree straight segments each, with kink angles of 88to 368 in-between (Table 2). Having kinks allows thetilted transmembrane helices to pack more inti-mately than otherwise possible and to enfold thechromophore in a tight binding pocket. The kinks inH1, H4, H5, H6 and H7 are caused by proline

1–30/TL

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T



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UNCORREC

residues at positions 53, 170 171, 215, 267 and 302,respectively. Except for Pro53 these proline residuesare among the most conserved residues inrhodopsin-like GPCRs,1,49 so this set of kinks isexpected at equivalent sequence positions through-out this family, although the kink angles may varyin a sequence-dependent manner. On the otherhand, H2 is distorted at the consecutive glycineresidues Gly89-Gly90, which allows the side-chainof Thr92 to H-bond to the main chain at residue 88.In H3 the kinks occur at Glu113, the counterion50–52

for the protonated Schiff base in the dark state, andnext to the ionone ring of the chromophore. In H7 asecond kink occurs near Lys296, which is at the startof the 310 segment and the site of chromophoreattachment. These kinks in H2, H3 and H7 are incontact with the chromophore and would be sharedby the visual pigments. Helix kinks have been notedin rhodopsin structures determined in the tetra-gonal form of 3D crystals and in the p22121 form of2D crystals;10,18 our definition (Table 2) differsslightly from earlier descriptions regarding locationand angle of the kinks.

The kinks are also important as sites of lateralpolar interactions, where rotation of the peptideplanes enables formation of inter-helical H-bondsand water-mediated H-bonds. Thus the helicesare linked by three-dimensional H-bonding net-works,18,19 which in our refined structure are moreextensive, laterally and perpendicular to the mem-brane, than previously reported.

Figure 5 shows one of the H-bonding networksthat involves Asn55 at the kink in H1, and alsoAsp83 in H2, Gly120 in H3, Met257 and Trp265 inH6, Ser298, Ala299, Tyr301 and Asn302 in H7, andfour water molecules including the hitherto uni-dentified Wat10. This network extends from Trp265in the chromophore binding pocket viaWat10 all theway to the highly conserved NPxxY motif53

(Asn302-Tyr306 in bovine rhodopsin) in H7 closeto the cytoplasmic limit of the hydrophobic zone(see Figure 3(a)). Asn55 and Asp83 in this networkare respectively the most conserved residues in H1and H2 of rhodopsin-like GPCRs.54 Trp265, Ser298,Ala299 and Asn302 are all widely conserved acrossthis family,1 and Met257 is conserved in vertebrateopsins. The proximity and interactions of theseresidues are critical to the stability and function ofGPCRs and of rhodopsin in particular.

The H-bonds between Asn55, Asp83, betweenAsn55 and the peptide O of Ala299 in the kink of H7at the C terminus of its 310 segment, and the water-mediated H-bonds between Asp83 and Asn302 onthe other side of this H7 kink can be expected tostabilise the packing of H1, H2 and H7 in the darkstate. H-bonding between these conserved residuesunderlies the apparent interaction among corres-ponding residues in related GPCRs.55–59 Forexample, the thyrotropin-releasing hormonereceptor was completely inactivated by mutationto a non-H-bonding residue at the position of Asp83or at both positions of Asn55 and Asn302.55 Inseveral receptors an Asp-Asn pair at positions of


ED PROOF

Asp83 and Asn302 was required for activation ofG-proteins, but their positions can be interchangedwithout loss of this activity.57–59 The examplessuggest that different H-bonding interactions ofthese residues are required for stabilising the darkstate and for attaining the activated state.In bovine rhodopsin Asp83 is not in contact with

the chromophore (Figure 5), but its replacement byAsn causes a slight blue shift of the absorptionmaximum indicating an alteration of the Schiff baseenvironment.60 Low temperature Fourier transforminfrared (FTIR) difference spectroscopy showedstructural changes in two internal water moleculesand peptide groups during the conversion tobathorhodopsin,61 the first intermediate statefollowing photo-isomerisation of the retinal,which would have been reached within pico-seconds of light absorption had it occurred atphysiological temperature.3,62,63 The spectralchange due to one of the water molecules isabolished when the Glu113 counterion is replacedby Gln, but the change due to the second watermolecule persists with a frequency shift.61 The latterwas thought to be vibrationally linked to the Schiffbase region through the peptide backbone and at alocation affected by replacement of Asp83 andGly120.61,64 This interpretation is borne out by thewater-mediated H-bonds between Asp83 and thepeptide backbone of H3 and H7 (Figure 5), andWat12 can be identified as that second watermolecule. Other FTIR studies showed that Asp83is protonated in the dark state and remains so in thebiochemically active metarhodopsin II (MII) state,but it undergoes a change or increase of H-bondingon entering MII.60,65 Note that the internal watermolecules in the surrounding of Asp83 (Figure 5)could, by small movements, facilitate rapidexchange of H-bonds without requiring concertedmovement of the protein.Trp265 in this H-bonding network was one of

the first chromophore contacts identified.66,67 Itsside-chain is in a U-shaped bend formed by the11-cis-retinal and the side-chain of Lys296, with theindole plane perpendicular to the membrane and incontact with the ionone ring (Figures 5 and 6).Resonance Raman data suggest that its micro-environment becomes less hydrophobic withinpicoseconds of photon absorption, which wasattributed to changing interaction with the iononering.68 On MII formation UV absorbance changesalso indicate a weakening of the indole H-bond inTrp265 and Trp126.69 In the dark-state structure theside-chain of Trp265 is oriented with the NH grouppointing to the cytoplasmic side and forming anH-bond with Wat10 (Figure 5), which is locatedin-line with the NH group. The UV difference inMIImight indicate a net displacement of the Trp265side-chain towards the extracellular side.The newly identified water molecule Wat10 is in

close contact with the side-chain of Ala124 in H3(Figure 5). In rhodopsin-like GPCRs the residue atposition 124 is highly conserved, but the predomi-nant amino acid is Ser.1 From this we infer that

30/TL

ECTED PROOF

Figure 5.A transmembrane slice showingH-bonding networks and hydrophobic contacts between the retinal-bindingpocket and the cytoplasmic surface.



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UNCORRWat10 is a surrogate for the Ser OH in providing anH-bond to the indole NH of Trp265, and further-more that H-bonding between this pair of side-chains in H3 and H6 is a conserved feature of GPCRstructure in the inactive state.

The side-chain of Ser298 is adjacent to Trp265, butit is H-bonded instead to the main chain O atresidue 295 (Figure 5). This side-chain to main chainH-bond compensates for the elongation of helixpitch at the start of the 310 segment (residues296–299). In rhodopsin-like GPCRs, position 298 ishighly conserved as Ser or Asn,1 both beingresidues capable of forming side-chain to mainchain H-bonds. Therefore it is likely, that H7 in thewider family also contains a 310 segment at this levelin the membrane.


Asp83 and Trp265 are both linked by H-bondsthrough ordered water molecules to Asn302 andMet257. This extended H-bonding network issealed from the cytoplasmic compartment by ahydrophobic barrier made of six residues fromhelices H2 (Leu76, Leu79), H3 (Leu128, Leu131) andH6 (Met253, Met257). Five of those residues arehighly conserved in rhodopsin-like GPCRs,1 andthe sixth, Met253, is 100% conserved in vertebratevisual pigments.49 They are arranged with meth-ionine residues on one side and leucine residues onthe other (Figure 5) and could facilitate the relativemovement of H3 and H64 following photoactiva-tion while retaining van der Waals contacts.Replacement of Met257 by all other amino acidsexcept Leu caused bovine opsin to activate Gt

1–30/TL

T

Figure 6. Environment of the 11-cis-retinal. (a) Environment of the protonated Schiff base and its counterion Glu113. (b)Environment of the ionone ring and the kinks in H6 and H7 cross-linked by a water molecule.



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UNCORREC

constitutively. The level of constitutive activity iswell-correlated with the affinity of mutant opsinsfor all-trans-retinal, suggesting that the mutationsdisposed the apoprotein towards the activeconformation normally attained only followinglight-induced chromophore isomerisation.70 Byimplication, Met257 in wt rhodopsin contributes acritical packing interaction. It is at the junctionbetween the H-bonding network and thehydrophobic barrier, is in contact with Asn302 ofthe NPxxY motif in H7, and prevents the helixmovement until this is triggered by chromophoreisomerisation.

Tyr306, at the cytoplasmic end of the NPxxYmotif in H7, is at the height of the lipid headgrouplayer (Figure 3(a)) where it is H-bonded via Wat7 toThr62 in H1 and directly to Asn73 in H2 (Figure 5),two residues which are conserved in vertebratevisual pigments and rhodopsin-like GPCRs,respectively. Tyr306 is stacked against Phe313 inH8, and similarly Ile307 in H7 is in contact withMet317. Such interactions can relay movements oftransmembrane helices to parts of the cytoplasmicsurface, for example realigning H8 after thephotoisomerisation. Spin-label data on Tyr306,Phe313 and Cys316 showed light-dark structuraldifferences,71 and double spin labelling showedmovements by 2–4 A of the cytoplasmic end of H7relative to that of H1,72 and of H2 relative to H8.73

The proper realignment of H8 appears tobe important in the interaction between the cyto-plasmic surface of rhodopsin and the C-terminalpeptide of Gta, because the inhibitory mutationseither map to the structurally sensitive connectingregion between the transmembrane H7 andperipheral H8 (Asn310, Lys311 and Gln312), orform buried contacts between H7 and H8 (Tyr306,Ile307, Phe313 and Met317).39,74–77


ED PROOFThe kinks in H3 and H5 are intimately associated

with the lining of the chromophore pocket. AtGlu113 in H3, a water molecule (Wat16) isH-bonded between the peptide carbonyl and side-chain carboxyl oxygen atoms. Consequently, thepeptide oxygen of Glu113 is not H-bonded alongthe helix axis, hence the kink (Figure 6(a)). Wat16 ismost likely the water molecule responsible for thatlow temperature FTIR difference feature betweenthe dark and batho states, that was abolished whenGlu113 was replaced by Gln.61

OE1 of Glu113 is H-bonded to the peptide N ofCys187 in b4 (Figure 6(a)), so the Schiff basecounterion is in contact with an H-bonding networkthat leads from the chromophore-binding pocket,through the plug, to the extracellular surface. Thisnetwork encompasses Tyr268 in H6, and residuesGlu181, Gly182, Gln184, Tyr191 and Tyr192 from theb3-b4 and b4-H5 loops (Figure 6(a)), as well as Tyr10from b2 and five water molecules. The H-bonds thatcross-link the b1-b2 and b3-b4 hairpins (Figure 4)belong to this network. Tyr268, whose OH group is3.6 A from the 11,12-cis bond of the retinal in thedark state (see Table 4 below) was implicated byFTIR and Raman studies to participate in the initialconformational change at the batho stage and in thetransition to the MII state.68,78,79

The second kink in H3 is in contact with theionone ring of the chromophore, where H3 and H5are linked due to a triangular set of H-bonds onthe side of the ionone ring away from Trp265(Figure 6(b)). The Trp126 indole NH forms a pair ofbifurcated H-bonds with OE1 of Glul122 and ND1of His211, while OE2 of Glu122 is H-bonded to thebackbone O of His211, which is so oriented becauseof the kink in H5 at His211 due to Pro215. Glu122 isknown to be protonated (neutral) in the dark stateand in MII.60 These interactions account for the

30/TL

T P

ROOFFigure 7. The non-conventional H-bond between a ring hydrogen of Phe203 in H5 and the backbone O atom in H4.

(a) Electron density map around Phe203. (b) Hydrogen bonds between residues in H4, H5 and the several loopsconnecting these helices and the b4, b5 strands. Four newly identified water molecules are located in this region. BesidesWat8 and Wat11 shown here, Wat14 is H-bonded to Pro194 O, Glu196 OE1 and Glu201 N, and Wat17 is H-bonded toTyr192 O and to Gly280 N in the H6-H7 loop. For clarity, the side-chains of Tyr191 and Tyr192 have been omitted.Figures 7(a), 8(a) and 10 were prepared using O.48



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UNCORRECFTIR observation that mutations of His211 influenceGlu122 through a third residue, which is itselfaffected by mutation of Glu122 to Ala but not toGln.80 Figure 6(b) suggests that Trp126 is the thirdresidue. These residues can be expected to showsensitivity to movements of the ionone ring, withwhich Glu122 is in direct contact. FTIR differencesuggested stronger H-bonding of Glu122 on tran-sition to MI,80,81 whereas UV absorption differenceindicated reduced hydrophobicity of the microen-vironment for Trp126 as well as weaker H-bondingof its indole NH caused by a general conformationalchange in the formation of MII.69

The kinks in H6 and H7 are only 4.8 A apartbetween Ca atoms of Tyr268 and Pro291. A watermolecule (Wat1), H-bonded to the backbone atTyr268, Pro291 and Ala295, bridges the kinks(Figure 6(b)). H6 shows the most pronounced kinkamong the rhodopsin helices (Table 2) and containsthe conserved Pro267-Tyr268 pair at this kink.Similarity in the light versus dark FTIR differencebetween [2H]tyrosine-labelled rhodopsin andbacteriorhodopsin has led to the proposal,79 thatduring photoactivation Pro267-Tyr268 in H6of rhodopsin may play an analogous role to


EDTyr185-Pro186 in helix F of bacteriorhodopsin.82

That is, to provide a pivot for the outward tilt of thecytoplasmic segment of H6 during the transition toMII as indicated by spin-label studies.4 The struc-tures of dark-state bacteriorhodopsin and a triplemutant mimic of its M-state (analogous to the MIIstate in rhodopsin) were shown to differ mainly byan outward tilt of the mutant’s helix F by amaximum of 3.5 A at the cytoplasmic end, whichcan be described as a hinge movement extrapolat-ing to a pivot around the Tyr-Pro pair.82 If a similarmovement occurs in the transition of rhodopsin toits MII state, then the water molecule between theH6 and H7 kinks would appear to be securing thepotential pivot in the configuration of the dark state.

At the kink in H4 near its extracellular end, the2FoKFc map (Figure 7(a)) reveals a non-conven-tional H-bond, with an aromatic ring hydrogenatom of Phe203 in H5 acting as donor83 to thecarbonyl O atom of Cys167 as acceptor (Figure 7(b)).The donor-acceptor distance of 3.10 A indicates arelatively strong H-bond. Residue 203 is Tyr in theconsensus sequence of rhodopsin-like GPCRs, andit is Tyr in 76% of the visual pigment sequences butPhe in the rest84 such as bovine rhodopsin. While

1–30/TL

T

Table 3. Dihedral angles along the conjugated double bond system of the 11-cis-retinal chromophore

Observed values (deg.) Refinement restraints

Dihedral angle Molecule A Molecule B Average (errora) (deg.) Target (deg.) Weight (kcal/mol)

C4, C5, C6, C7 K179 177 179 (2) 180 100C5, C6, C7, C8 K57 K53 K55 (2) None noneC6, C7, C8, C9 K179 K176 K178 (2) 180 100C7, C8, C9, C10 K178 K178 K178 (0) 180 100C8, C9, C10, C11 179 179 179 (0) 180 100C9, C10, C11, C12 176 173 175 (2) 180 100C10, C11, C12, C13 K10 K15 K13 (2) 0 50C11, C12, C13, C14 173 170 172 (2) 180 50C12, C13, C14, C15 176 176 176 (0) 180 100C13, C14, C15, Nx 177 178 178 (1) 180 100C14, C15, Nx, C3 179 K179 179 (0) 180 100

a Error: deviation of observed values from their mean in the absence of noncrystallographic symmetry restraints on the retinal moiety.



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UNCORREC

Tyr can be expected to form an H-bond at thisposition, our observation demonstrates that in thelow dielectric medium of the membrane interior,Phe can replace Tyr in donating a stabilisingH-bond to the conserved kink in H4.

From the Pro170 at the kink in H4 to Asp190 atthe end of the b3-b4 hairpin, visual pigmentscontain nine highly conserved residues, attestingto the importance of this region to the structure andfunction of rhodopsin.84 Figure 7(b) shows some ofthe H-bonds from the conserved Pro170, Pro171,Gly174, Trp175, Tyr178 and Asp190, which fastenthe b-hairpin to the helix bundle. The hairpin isanchored to H3 by the disulphide bond betweenCys110 and Cys187, and its first and last residues,Arg177 and Asp190, form a salt bridge (Figure 7(b))whose removal causes release of retinal fromrhodopsin in the dark without apparentlyincreasing the accessibility of the Schiff base tobulk solvent.85 This destabilisation is probablydue to perturbation of the H-bonding network(Figure 6(b)) that helps to position b4 at the “floor”of the retinal-binding pocket.

Conformation and environment of thechromophore

Table 3 lists the torsion angles along theconjugated double bonds of the 11-cis-retinal andits imine linkage to Lys296. Uncertainties wereestimated by comparing values in the two NCScopies after one cycle of mock refinement in theabsence of NCS restraints on the retinal moiety. Thechromophore is confirmed as containing threeplanar segments,20 with deviations from theplanarity of the p-bonding system about the 6s-cis,and 11-cis, 12s-trans bonds because of the stericinteractions between 5-CH3 and 8-H, and between13-CH3 and 10-H, respectively. The conformation ofthe chromophore in our structure is similar to thatin the 1F88 coordinates for the tetragonal crystalform,14 and disagrees with the 1HZX18 and 1L9H19

coordinates. For example 1L9H shows a planar11-cis bond but significantly non-planar C8–C9 andC14–C15 bonds. It should be pointed out that whenwe used the 1F88 coordinates for the molecular


ED PROOF

replacement model, we excluded the retinal as a testagainst model bias, and we have built the initialmodel of the chromophore from the small moleculecrystal structure of 11-cis-retinal20 by adjusting itstorsion angles to fit the cross-crystal averagedelectron density map (see Methods), which wasconsistent with three planar segments. Refinementwas carried out with planarity restraints imposedon the chromophore, as was appropriate at themedium resolution. However, the weights of therestraints (Table 3) were set to be 5–12 times smallerthan those on peptide bonds or aromatic rings, sothey could not have induced a planar conformationhad it not been rooted in the X-ray data. Thus ourstructure provides a credible description of theretinal-protein interactions in dark state.The torsion angle about the 11-cis bond is

determined to be K13(G2)8 (Table 3 and Figure 6).This value is compatible with the calculation, basedon analysis of resonance Raman intensities, that theC11]C12 torsion angle increases, within 50 fs afterexcitation, from K158 in the dark state towardsthe 908 transition state for the cis-to-transisomerisation.86 Both the rapidity and stereospeci-ficity of the initial torsional dynamics are driven bythe strain on this non-planar segment.86,87 Thechirality and pucker of the ionone ring are also inagreement with NMR data.88

Figure 8(a) shows the electron density maparound the retinal. Figure 8(b) is a schematicdiagram of the retinal-binding pocket, showingH-bonds of Lys296 and Glu113, and the mostlyhydrophobic contacts with the retinal within adistance of 4 A. Those contact distances are given inTable 4. The elongated 11-cis-retinal buries about220 A2 of surface area on a total of 22 residues thatform the binding pocket. Roughly one third of thisarea is contributed by Trp265 and Tyr268 in H6,another third is from Thr118, Glu122 in H3 andMet207, Phe212 in H5, and the rest is made up by 16residues from helices H3 to H7 and the plug14

(Ala117, Lys296, Cys187, Phe261, Ala269, Phe208,Ala292, Tyr191, Gly121, His211, Gly188, Cys167,Ile189, Glu181, Ser186 and Ala272 in descendingorder of contact area). The large number ofcontact residues in addition to its covalent linkage

30/TL

UNCORRECTED PROOF

Figure 8. The retinal-binding pocket. (a) Electron density map around the 11-cis-retinal. (b) Schematic drawing of thebinding pocket showing all residues within 4 A of the retinal and Lys296. This drawing was modified from the output ofLIGPLOT.148




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2016

UNCORRECTED PROOF

Table

4.Retinal

contact

distances

Contactsupto

4A

Chromophore

atoms

Protein

atoms

C2

C3

C4

C5

C6

C8

C9

C10

C11

C12

C13

C14

C15

C16

C17

C18

C19

C20

NZ

K296

H1

Met

44CE

3.7

SD

3.7

Leu

47CD1

3.8

H2

Phe

91CE1

3.9

CE1

4CZ

3.6

H3

Glu

113

CD

3.9

OE1

3.8

3.7

OE2

3.2

3.9

Ala

117

CB

3.6

3.7

3.7

Thr

118

OG1

3.6

3.8

3.7

3.2

CG2

3.9

Gly

121

CA

3.5

Glu

122

N3.8

OE2

3.6

3.4

3.8

3.8

b3-b4

Ser

186

CB

3.9

3.4

OG

4.0

b4

Cys

187

O3.6

3.0

3.9

3.8

Gly

188

CA

3.7

3.9

Ile

189

N4.0

CG1

3.5

b4-H5

Tyr

191

OH

3.4

H5

Met

207

CG

3.6

SD

3.6

CE

3.5

Phe

212

CB

4.0

CG

3.8

CD1

3.3

3.5

CE1

4.0

3.9

H6

Phe

261

CZ

3.9

3.5

Trp

265

CD2

4.0

CE3

3.9

3.7

CZ2

3.9

CZ3

3.7

3.6

3.9

CH2

3.7

Tyr

268

CD2

3.9

CE2

3.6

3.6

3.5

CZ

3.8

OH

3.7

3.6

4.0

H7

Ala

292

O3.5

3.8

3.9

C4.0

Phe

293

CA

3.9

CG

3.8

CD1

4.0

CD2

4.0

CE2

4.0




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2120

2121

2122

2123

2124

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2126

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2128

2129

2130

2131

2132

2133

2134

2135

2136

2137

2138

2139

2140

2141

2142

T



2143

2144

2145

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2147

2148

2149

2150

2151

2152

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2157

2158

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2161

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2165

2166

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2168

2169

2170

2171

2172

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2174

2175

2176

2177

2178

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2181

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2240

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2242

2243

2244

2245

2246

2247

2248

2249

2250

2251

2252

2253

2254

2255

2256

2257

2258

2259

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2266

2267

2268

UNCORREC

contribute to a favourable binding enthalpy ofK11 kcal molK1.89 In our structure, the sevenresidues with the lowest B-factors, Trp265, Ala295,Ala269, Thr118, Phe91, Ala117 and Tyr268, areall located around the retinal-binding pocket(Figure 8), which attests to the stabilising inter-action between the chromophore and its bindingpocket. This stabilisation is responsible for the“inverse agonist” effect of the 11-cis-retinal thatkeeps the basal activity of rhodopsin towards Gt inthe dark very low, below that of the ligand-freeopsin, which is itself 106-fold less active than theMII state with all-trans-retinal bound.90 The threearomatic residues showing the largest chromophorecontact areas, Trp265, Tyr268 and Phe212, are highlyconserved throughout rhodopsin-like GPCRs,1

suggesting common elements in the mechanismfor stabilising the inactive receptor conformationfrom the boundaries of the ligand-binding pocket.

Electrostatic interactions of the binding pocketwith the chromophore affect the visible absorptionmaximum.91–94 Three residues near the ionone ringcorrespond to the major colour tuning positions inred/green cone pigments identified by primategenetics.95 Among these Phe261 and Ala269 aredirect contacts of the ionone ring (Figure 6(b)), andmutagenesis to Tyr and Thr, respectively, repro-duced the predicted red shift,91 which is consistentwith a dipolar stabilisation of the positive chargeredistribution in the excited state chromophoretowards the ionone ring.94,96 The third residue,Ala164, is a second shell contact of the ionone ringvia Glu122. In rhodopsin its substitution by Serproduced only a minor red shift,91 but this can beexplained by the dipole contribution of Ser beingovershadowed by the intervening Glu122,97 sincereplacing Glu122 by Asp was known to cause a blueshift.66 In the red/green cone pigments where theconserved residue at the position 122 is Ile, a strongtuning effect can be expected.

The structure of the binding pocket is illustratedin Figure 6 with the Schiff base (Figure 6(a)) or theionone ring (Figure 6(b)) in the foreground. In theSchiff base region, the Lys296 side-chain is fullyextended and held in a channel between Met44,Leu47 and Phe91 from H1 and H2 and Phe293 fromH7, as previously reported14. The Glu113 side-chainis in an unusual rotamer because Wat16 isH-bonded between its peptide O and carboxylOE2 atoms. The OE2 atom is thus placed in-linewith the NH bond of the protonated Schiff base.Thr94 and Gly90 from H2 make close contact withGlu113 and Wat16 bound to it (see below). OE1 ofGlu113 is H-bonded to the backbone of Cys187,which is in a disulphide bond with Cys110 in H3.Thus the geometry of the Lys296, Glu113 ion pair isstringently defined. The structure suggests that asalt bridge between them can also exist in theapoprotein, where it is required for maintaining theinactive state.98

In Figure 8(b), Wat16 forms a shorter H-bond tothe negatively charged carboxyl OE2 than to theneutral peptide O, showing that it is strongly bound


ED PROOF

to the anion. The Wat16-mediated H-bondsdelocalise the negative charge on the Glu113carboxylate and lower its pKa. Stabilisation of theanion, aided by the precise geometry of the ion pair,in turn stabilises the proton on the Schiff base,elevating its pKa in the dark state to O16.99

Therefore spontaneous deprotonation is kept at alow rate, which is essential for dim light vision.100

Change of geometry in the Schiff base region afterthe photoisomerisation can destabilise the protonand pave the way for the deprotonation of Schiffbase in MII.

The distance between OE2 of Glu113 and theSchiff base nitrogen is 3.2 A (Table 4) on both NCScopies, and is in agreement with the 3.1 A distancein the P41 crystal form.19 This is less than the 4.3 Adistance implied by a similarity of 15N chemicalshift of the protonated Schiff base in rhodopsinand that in model retinylidene with iodide ascounterion.101 However delocalisation of thenegative charge in Glu113 effectively movesthe centre of the counterion further away fromthe protonated Schiff base by about 1 A, thusweakening their electrostatic coupling to permitgreater delocalisation of the positive charge fromthe Schiff base into the polyene of the retinal,102

which was indicated by the nitrogen chemical shift.Therefore, Glu113 and Wat16 form a complexcounterion in the dark state, which both stabilisesthe proton on the Schiff base and determines thewavelength of the visible absorption maximum.That mutating Glu113 to the smaller Asp results in ared shift without dependence on external chlorideions is also consistent with an increase of the ionpair distance.103

Recently because the Glu181 to Gln mutation wasfound to lower the pKa of the Schiff base underMI-like conditions, the counterion was proposed toswitch in MI to Glu181,104,105 accompanied byreorganisation of the E2 loop coupled to movementsof H3.105 The dark state structure suggests that theproposed backbone movements are not required forthe Glu181 side-chain to get close enough to theSchiff base to measurably influence its electrostaticenvironment. The Schiff base NH bond in theall-trans retinal may be tilted more towards Ser186and Glu181 on the H6, H7 side of the polyenebackbone (out of the page in Figure 6(a)), instead oftilting towards Glu113 on the H3 side as in the darkstate structure.106 The NMR result, that the iononering and the adjacent segment of the retinal in MIdespite being more relaxed remain largely in thedark state conformation,107 is consistent with theretinal isomerisation up to this stage involvingrotations mainly on the Schiff base side of theisomerised bond. There is also evidence that theretinal isomerisation impinges on Tyr268.68,78 Itseffect on the H-bonding network (Figure 6(b)) mightallow the Glu181 side-chain to rotate towards theSchiff base. Such localised rearrangements fromthe known dark state structure may apply to theinvertebrate rhodopsins, which lack an acidicresidue at position 113 and use a conserved residue

1–30/TL

UNCORRECTED PROOF

Figure 9. The cytoplasmic domain. (a) Residues implicated in interaction with the heterotrimeric G protein transducin.The main chain is coloured in the four segments that project above the lipid phosphate groups and form the cytoplasmicdomain: C1 loop region (residues 64–71), C2 loop region (134–152), C3 loop region (225–252), and H8 and C-terminal tail(310–326). The side-chain colours indicate: green, mutations causing constitutive Gt activation; red, residues cross-linkedto Gta; pink, mutations inhibiting stabilisation of MII by Gta 340–350; yellow, mutations reducing Gt activation; cream,gain-of-function for Gt activation upon reverse mutation from Ala; blue, Arg135 of the conserved E(D)RY motif; grey,other residues discussed in the text. Residues falling in more than one category are coloured according to the formercategory. Long broken lines joining pairs of Ca atoms marked by grey spheres indicate inter-helical cross-links thatinhibit activation.35,119 The footprint of the 11-cis-retinal in the membrane plane (light pink) is shown in the backgroundto illustrate its vertical alignment with key residues on the cytoplasmic surface. (b) Topography of the cytoplasmicsurface showing a cavity adjacent to H3, with Leu131 at its bottom, and the Glu134-Arg135 pair forming a salt bridge inits walls. Note the close contact of Arg135 with H6. This Figure was prepared using PYMOL.




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CORREC
corresponding to Glu181 as the counterion,108 andto uv-absorbing pigments, which have a neutralGlu113 in the dark state and switch to Glu181 forthe counterion in MI.109,110

Steric interactions with the binding pocket allowrelaxation of the photo-isomerised retinal to triggerconformational changes that result in Gt activationin MII. The ionone ring is under steric restraintsfrom residues in helices H3, H5 and H6 (Table 4;Figure 6(b)) up to the MI state.107 Evidence for achange in the microenvironment of adjacentTrp26569 in the formation of MII may indicate amovement of the ionone ring. A ringmovement willimpinge on Trp265, which is the most orderedresidue in the dark state structure, the residue withthe largest contact area with the retinal, and iswidely conserved among GPCRs. It may also affectPhe261, a conserved aromatic residue in visualpigments which is in contact with the C3, C4 edge ofthe ring and located in the cytoplasmic segment ofH6 one helical turn above Trp265 and one belowMet257 (Figure 5). The residue at position 261 in theglycoprotein hormone receptors has been shown toplay a similar role to Met257 in rhodopsin informing a stabilising helix packing interactionwith Asn302 in H7.70,111 Thus retinal interactionswith the aromatic residues following photo-isomerisation could precipitate a release of thepacking interactions between H3 and H6 (seeprevious section on Transmembrane domain), andbetween H6 and H7,70 that stabilise the inactivestate. As shown in Figure 5, and in Figure 9(a)below, when the dark state structure is placed in themembrane frame, the ionone ring appears verticallybelow the conserved Glu134, Arg135 charge pair atthe cytoplasmic border of H3, where light-inducedprotonation of Glu134 is a prerequisite forrhodopsin to bind Gt.112–114 The H-bondingnetworks and van der Waals contacts described inthe transmembrane domain serve as conduits forvectorial transmission of the conformationalchanges across this distance, in details that arestill unknown. Deletion of the 19-CH3 attached toC9 of the polyene chain was shown to result in alooser fit of the chromophore analogue in thebinding pocket, consequently reducing the entropygain115 that drives the conversion from MI to MII.89

Therefore the 19-CH3 in the native structure,wedged between Thr118 from H3, and Ile189 andTyr191 from the plug (Figure 6(a)), appears toprovide a grip against the floor of the bindingpocket and restrict the relaxation of the chromo-phore so that it is directed into a productiveactivation. Thus steric interactions of all parts ofthe chromophore are important for the activation.

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UNCytoplasmic domain

Having positioned the X-ray coordinates in themembrane frame (Figure 3(a)), we regard allresidues above the mean level of phosphate groups(beyond 20 A from the mid-plane of the bilayer) asbelonging to the cytoplasmic domain. They include


the three cytoplasmic loops (C1, C2, C3) andadjoining ends of transmembrane helices (residuesGln64-Pro71, Glu134-His152 and Gln225-Arg252),the peripheral helix H8 and adjoining loops(residues Asn310-Asn326), and a di-peptidefragment from the C-terminal tail (Figure 9).

ED PROOF

C1 loop and C-terminal fragment

The C1 loop (residues 65–69) is very similar tothat in the P41 form.14 It is well ordered (Figure 3(a))and comprises basic and hydrophobic residues(His65, Lys66, Lys67, Leu68, Arg69), which exceptfor His65 are more than 97% conserved amongvertebrate opsins, more conserved than the ends ofH1 and H2. Leu68 and Arg69 point inward, makingvan der Waals and H-bonding contacts withresidues in H1, H2 and H8; the other residuespoint outward to form a basic patch. The 2FoKFcmap next to the C1 loop shows a strong densityfeature with the flatness of a peptide plane incontact with the side-chains of His65 and Lys67 andthe main chain from His65 to Lys67. Its aqueouslocation and shortness make it rather unlikely tobelong to a detergent molecule. We have interpretedthis density as a di-peptide from the disorderedC-terminal tail and tentatively assigned it to Asp330and Asp331, because of the basic contact site andbecause the distance from the last ordered residue,Asn326, can be spanned by three or more residueswith reasonable main chain torsion angles. The Cterminus of the putative di-peptide points towardH6. No other interpretable density was found forthe C-terminal tail in the P31 crystal form. Thepresence of a thermolysin cleavage site betweenPro327 and Leu328116 and an Asp-N endoprotei-nase cleavage site between Gly329 and Asp33016 isconsistent with solvent exposure of the unobserved327–329 segment. Site-directed spin labelling ofrhodopsin in dodecylmaltoside micelles showedthe C-terminal tail to be in equilibrium between anordered and disordered conformation, with thedisordered component increasing dramaticallybeyond Asp331.34 The density adjacent to loop C1probably corresponds to the partially orderedcomponent. In the P41 structure loop C1 alsoforms contact with the C-terminal tail, but thechain orientations (Figure 2(b)) and residue assign-ments differ. Our tentative assignment of Asp330and Asp331 pointing towards H6 is reconcilablewith the report that the Cys mutant at position 338in the C-terminal tail does not form a disulphidewith the Cys mutant at position 65 in the C1 loop,but it does cross-link with Cys mutants at positions242, 245 and 246 in H6, rapidly in the dark andslowly in the light.117

C2 loop and cytoplasmic ends of H3 and H4

The C2 loop (residues 141–149) is L-shapedin both crystal forms, but lies more parallel tothe membrane surface in the present structure(Figure 2(b)). It is as if the two conformations

1–30/TL

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UNCORREC

were related through a hinge movement, andcomparisons with the NCS differences showedthat it is most likely caused by different packingcontacts in the two crystal forms. The coordinatedifferences are above background level fromresidue 136 to 150 (Figure 2(a)), starting at thepoint where the highly tilted H3 becomes accessiblefrom the surface (Figure 9). Thus the cytoplasmic tipof H3 appears to bend with C2 as the loop isre-oriented. The middle of C2, where B-factorsexceed 100 A2 in both crystal forms, is probablyrather flexible.

Peptides corresponding to the C2 and C3 loops,and H8, have been shown to interact with Gt.118

Residues important for Gt activation (Figure 9(a))are found in the C terminus of H3 and in the C2loop. Glu134, Arg135, Tyr136 form the highlyconserved E(D)RY motif found in H3 of allrhodopsin-like GPCRs. In the dark state, Glu134 isnegatively charged and forms a salt bridge withArg135. The latter faces H6 and forms van derWaals contacts with Val250, Thr251 and Val254 inH6. Protonation of Glu134, in addition to deproto-nation of the Schiff base, is required for rhodopsinto activate Gt.114 The cytoplasmic surface ofrhodopsin shows a cavity next to H3 (Figure 9(b)).Leu131 from the hydrophobic cluster that seals theinter-helical H-bonding network (Figure 5) fromthe cytoplasm is visible from the opening. But thecavity extends like a tunnel under Pro71 andArg147 to reach Phe148 and Ala153, and mightprovide a site for transducin to interact withrhodopsin. The Glu134, Arg135 charge pair issequestered in the wall of this cavity in the darkstate, so that they can form a salt bridge, but Glu134also has access to the aqueous phase to take up aproton. Protonation is thought to cause Glu134 tomove to a non-polar environment and favoursbinding to Gt.113 Loss of the salt bridge partnermight also cause Arg135 to transfer to the aqueousphase, thus breaking its van derWaals contacts withthe residues along H6 (Figure 9(a)). This wouldfavour a parting of the cytoplasmic ends of H3 andH6, which is believed to occur in the activation ofrhodopsin as indicated by studies using spinlabelling,4 metal chelation,119 and disulphidecross-linking (Figure 9(a)).35 The Gln134 mutant,which mimics the protonation, favours binding ofrhodopsin to Gt113 and causes constitutive activityof opsin.112 This mutant shows increased mobilityof spin label attached to Cys140 in H3 or Cys316 inH8 in the dark,120 which is otherwise associatedwith photoactivation.121–123 Eliminating the chargeon Arg135, the other half of the salt bridge, does notcause the same mobility change.120

The binding of Gt or the C-terminal peptide ofits a-subunit (Gta 340–350) stabilises MII.124,125

Replacing the Tyr136-Val139 sequence reducedGt activation by w80% and abolished stabilisationof MII by Gta 340–350.28 Tyr136, which pointsoutwards and does not interact with the rest ofthe protein, is an important determinant forthe stabilisation of MII by Gta 340–350, but


Val137-Val139 may also contribute stabilising inter-actions for Gt binding.28 Val138 is adjacentto Glu134 and Arg135 (Figure 9(a)). Mutatingindividual residues of Tyr136-Val139 to cysteinealso caused partial loss of Gt activation.27 Spin labelattached to Lys140 showed rise and fall of mobilitywith time constants corresponding to the formationand decay of the MII state.122 Therefore thecytoplasmic end of H3 is involved in the formationof the active state and binding of Gt.In the C2 loop (residues 141–149), mutating

Lys141 to Cys reduced Gt activation.126 Reagentsattached to this residue cross-linked photoactivatedrhodopsin to Gta.38 Replacing the sequenceLys141-Arg147 caused partial loss of Gt activationbut did not prevent binding of Gta 340–350.28 Aftermutating Lys141-Phe148 to polyalanine abolishedactivation, restoring only two residues from eachend, namely Cys140, Lys141, Arg147 and Phe148,restored 50% of the activity, but the middle residuesalso contribute to activation, possibly by influen-cing the conformation of the loop.39 The flexiblemiddle section might become more ordered whenMII binds to Gt and help to increase the affinity ofGt binding.

ED PROOC3 loop and cytoplasmic ends of H5 and H6

Helices H5 (residues 200–230) and H6 (residues241–276) in our structure are both longer byone helical turn at the cytoplasmic boundarythan previously reported. Therefore the C3 loop iselevated above the membrane surface (Figure 2(b)).This loop has the highest B-factors in the wholerhodopsin molecule (Figure 3(a)). However theseB-factors are authentic indicators of main chainflexibilities, since the relative scale of B-factors inthe C3 loop compared to those in the C2 loop issimilar to the relative nobilities of these surfaceloops measured by spin-label studies in detergentmicelles.43 During model building, main chainfragments selected from a data base of well-refinedhigh resolution structures by the program O48 fortheir similarity to the densities of the cytoplasmicends of H5 and H6 and the loop were tightlyclustered and all showed helix-like sections. Afterrefinement with the best fitting fragment insertedinto the C3 loop density, the real space correlationwas 0.72 in the C3 loop whereas the average for thewhole structure was 0.95. The density was poor forresidues Ala233-Ala235 and Gln238, which have thehighest B-factors. So, coordinates for the C3 looprepresent the most probable conformation in apopulation of overlapping, variable conformations.However the extra helical turn at the ends of H5 andH6 compared with the P41 coordinates isunambiguous.From residue 231 to 236 the C3 loop rises from the

membrane like an extension of H5, but with the axistilted from H5 towards the cytoplasmic tip of H3;from residue 236 onwards, the loop follows anotherspiral path running parallel to the membrane untilit joins H6. The tip of H6 from residue 244 to 241 is

30/TL

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ORREC
bent towards H5 to meet with the loop. Thesecondary structure of loop C3 consists ofhydrogen-bonded turns and not regular a-helices,33

but the periodicity of accessibilities detected by aprevious spin-label study33 is accounted for.Therefore the P31 structure and the spin labelstudy both show the C3 loop to be well elevatedfrom the bilayer and variable but not unstructured.

Residues around the C3 loop that were shown bymutagenesis to affect Gt activation map to thecytoplasmic end of H5 (Leu226, Thr229 andVal230)127 and the first turn of H6 (Thr242, Thr243and Gln244),39,127 which are ordered in the darkstate, and to the spiral extension of H5 (Ala233,Ala234),29,127 which is highly mobile. All threeregions project well above the cytoplasmic surfacein our structure (Figures 2(b) and 3(a)). Ser240 nextto the N terminus of H6 is the residue most elevatedabove the membrane. Reagents attached to theSer240Cys side-chain cross-linked the light-activated rhodopsin mutant to Gta.37,38,128 Thecross-linked sites were located within the Gtapeptides 19–28,38 310–313 and 342–345,128 whichoverlap respectively with the three rhodopsin-binding regions of Gta mapped with syntheticpeptides, 8–23, 311–323 and 340–350.125 Themutants Lys141Cys and Lys248Cys can also becross-linked to Gta.38 Replacing Glu247-Thr251 inH6 by unrelated sequence reduced Gt activationtenfold and abolished Gta 340–350 binding.28

Restoring the sequence of Glu247-Glu249 restoredGta 340–350 binding, but only part of the Gtactivation.28 Tyr136-Val139 and Glu247-Glu249were proposed to form a binding pocket for Gta340–350.28 In the dark state structure they map toH3 and H6 at similar heights from the membrane(Figure 3(a)). The effects of the mutations may haveresulted from interactions with other sites, sinceVal250 and Thr251 are in contact with Arg135 in H3,Thr251 with Leu226 in H5 (Figure 9(a)), and Glu249with residues in H7 and H8 (see next subsection onH8). In chimeras of the Gt activating bovinerhodopsin and Go activating scallop rhodopsin,replacing the C2 loop with the scallop sequencereduced activation of both G-proteins, but replacingthe C3 loop with the scallop sequence enhancedactivation of Go fourfold while depressing acti-vation of Gt by 60%.129 Therefore the C3 loopcontains sites for specific activation of G-protein.The ability of the C3 loop of bovine rhodopsin todiscriminate between different Gta 340–345sequences may be amechanism to ensure specificityof coupling.129

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UNCH8 and adjoining loops

The structure of H8 (residues 311–321) is verysimilar in both crystal forms. Asn310 is the onlynon-helical residue between H7 and H8. Severalside-chain to main chain H-bonds stabilise theH7/H8 corner, such as from Glu249 to Met309and Lys311, from Asn310 to Phe313 and fromArg314 to Ile307. In addition, H8 is anchored at


the membrane–aqueous interface by buried side-chain contacts with residues in H1 and H7 under-neath, such as the contacts of Phe313 with Thr58,Val61, Thr62 and Tyr306, and of Met317 with Leu57,Val61 and Ile307. The presence of Asn310 andLys311 are required for correct folding of therhodopsin.39 If these two residues are kept, thenreplacing all other residues in H8 except Phe313and Met317 by Ala, including replacing all outwardpointing residues, produced amutant pigment witha potency for Gt activation equal to the wild-typerhodopsin.39 The contacts of these four residues justdescribed suggest that they are required for thecorrect tertiary structure and orientation of H8.Deleterious effects of mutations in the N-terminalpart of H8 are all interpretable in terms ofperturbation of the interaction between H8 andthe transmembrane domain.75–77 It has been shownthat truncating rhodopsin following Asn315 doesnot diminish Gt activation.130 Therefore althoughH8 participates in binding of Gt, its role inactivation of Gt is secondary to the C2 and C3loop regions.

ED PROOFExpected location of G-protein interaction sites in

other GPCRs

In the C2 loop, residues important to activationhave been mapped in the conserved E(D)RY motifor just C-terminal to this. The C3 loop varies widelyin length among GPCRs. As seen in the bovinerhodopsin structure, however, residues believed toparticipate in Gt binding and activation are clus-tered around the cytoplasmic ends of H5 and H6and the spiral extension immediately above H5,while residues shown by alanine scanning muta-genesis to have no effect on Gt activation29 map tothe C-terminal section of C3, which runs across thegap between H5 and H6. We may expect that, in theC3 region of other GPCRs, the Gt interaction siteswill be similarly clustered with respect to thetransmembrane helices. In the cannabinoid recep-tor, the C3 loop is about 30 residues long and a smallhelix was detected by NMR adjacent to thecytoplasmic end of H5,131 which is analogous tothe H-bonded spiral extension of H5 in bovinerhodopsin, even though there is no sequencehomology in this region. In the muscarinic acetyl-choline receptors, the C3 loops are very large, butdeletions of over 100 residues to reduce it to thelength of C3 in bovine rhodopsin affected only thedesensitisation process and left the G-protein-dependent functions intact, such as agonist andantagonist binding and phospholipase acti-vation.132,133 Therefore it is likely that these residuesmapped in the C2 and C3 regions of bovinerhodopsin are the common G-protein-bindingsites in all GPCRs.

Bound lipid and detergent molecules

The presence of a bound phospholipid moleculenear the cytoplasmic ends of H6 and H7

1–30/TL

UNCORRECTED PROOF

Figure 10. Bound lipid and detergent molecules. (a) Two acyl chains of a phospholipid molecule bound to H6 and H7in the cytoplasmic half of rhodopsin. The cytoplasmic segment of H6 is orientated roughly along the membrane normal.The acyl chains are inclined relative to it. Met309 is the C-terminal residue of H7. (b) An ordered LDAOmolecule boundto H5, H6 and H7 just to the extracellular side of the kinks in H6 and H7. Pro267 is one of the contact residues. (c) A C8E4

molecule bound in the crevice between stacked helix bundles of the P31 crystals.




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(Figure 10(a)) is indicated by densities for a pairof acyl chains with a mean separation of4.15(G0.02) A, which is characteristic of closepacked hydrocarbon tails in an ordered bilayer.The lipid acyl chains form contacts with residues inH6, Ile256, Ala260 and Ile263 in molecule A andIle256, Ala260 in molecule B, and in H7, Thr297,Tyr301, Val304, Ile305, Met309 in molecule A andThr297, Ty301, Ile305 and Met309 in molecule B.The identity of this phospholipid is unknown,because no density was observed for the headgroup, which is located in the solvent channel and isapparently disordered.

The addition of 0.05% LDAO to C8E4 solubilisedrhodopsin was the key to obtaining isotropicallydiffracting crystals of this form.16 The structureshows a single ordered LDAOmolecule in additionto several partially ordered C8E4 molecules boundto each rhodopsin molecule. The LDAO is boundjust to the extracellular side of the H6 kink(Figure 10(b)) and is itself kinked at the C5 atom,with the amine oxide arm pointing roughly alongH6 towards the extracellular face and the hydro-carbon arm running from H6 towards H5. Thecontacts are with H6 mainly (Leu266, Pro267,Gly270, Val271, Phe273, Tyr274), but also withH5 (Phe208 and Phe212) and H7 (Phe287). Inwrapping around these helices, the LDAO may bere-enforcing the more ordered molecular structureoriginating from the extracellular leaflet (seeFigure 3(b)), therefore stabilising a particularprotein conformation resulting in better orderedcrystals. Pro267 in the binding site of LDAO hasbeen proposed as a possible hinge in H643,79 for theoutward tilt of its cytoplasmic segment uponphotoactivation.4,35,119 The presence of a hingemidway along H6 is supported by disulphidelinking experiments showing a lack of relativemovement between the extracellular ends of H6and H5 during activation.134 Note that in the darkstate structure H6 is kinked in a plane tangentialto the helix bundle. In the activated form thecytoplasmic segment of H6 is kinked about thesame place but in a radial plane instead. For thischange to occur, the structure of the hinge regionmay have to undergo a rotation first. Recently,cryo-EM of rhodopsin 2D crystals highly enrichedfor the MI state showed a local density change nextto the dark state position of Trp265,135 which wouldbe consistent with mobilisation of the hinge prior tothe larger conformational changes required to enterthe active MII state. Thus the LDAO moleculewrapped around the kink in H6 appears to stabilisethe inactive conformation around a potential hinge.

The most completely modelled C8E4 moleculeshows the octyl chain and three ordered oxyethy-lene groups out of the total four. The elongateddetergent molecule is intercalated in a taperedcrevice between rhodopsin helix bundles, as theyare stacked along the 31-screw axis. Together withthe palmitoylate chains linked to Cys322 andCys323 it fills this crevice (Figure 10(c)). Thereare hydrophobic-to-hydrophobic and hydrophilic-


to-hydrophilic contacts between the C8E4 and twoprotein molecules, one of them presenting thecytoplasmic end of H7 and H8 and the otherpresenting the extracellular end of H4 and H5(Figure 10(c)). The choice of C8E4 as the detergentand its interactions in this crevice most likelyprecipitated the P31 crystal form.

Methods

ED PROOF

Data collection and processing

Crystallisation and preparation of heavy atomderivatives are described in the accompanying paper.16

Complete data sets were obtained by combining wedgesof 20–30 degrees collected from different positions on oneor more crystals. Data were processed using programs inthe CCP4 suite.136 Intensities were integrated toanisotropic resolution limits using mosflm, and mergedusing scala. A consistent indexing regime was main-tained, by re-indexing each wedge under the fourindexing regimes of point group P3, and selecting theone leading to the lowest R-factor on amplitudes (Rderiv),calculated using scaleit, relative to a previously mergedreference data set or partial data set. Using the sameprocedure, data sets from non-isomorphous crystals wereplaced under a common indexing regime prior to cross-crystal averaging. The intensities were scaled in tworounds. The first round was done in the “batch” mode,with the batch showing the least negative B-factor servingas reference for B-factor normalisation; batches foundwith relative B-factors above 13, indicating significantradiation damage, were rejected from further scaling. Thesecond round used smooth scale and B-factors, in whichsecondary beam corrections in the form of sphericalharmonic functions were determined for each crystal.A “tails” correction for contributions from diffusescattering was applied during both batch and smoothscaling rounds. Twinning was detected by inspecting thecumulative intensity distribution given by truncate.When it was present, the twin fraction was estimatedand intensities were de-twinned using the programDetwin.136

Molecular replacement from the 1F88 coordinates andrefinement

Molecular replacement was done using the programamore137 and the rhodopsin coordinates in the P41 crystalform at 2.8 A resolution (PDB code 1F8814,17) as searchmodel. The two rhodopsin molecules in the P41asymmetric unit were superimposed to form one searchmodel, but only the more ordered and more complete ofthe two molecules (chain A) was used to calculate initialphases. Non-protein atoms were omitted from the searchmodel and initial phase calculation. In the calculatedmaps, the density for the retinal moiety, which wasomitted from the model, was used to assess the quality ofthe phasing.Molecular replacement was done in parallel against

each data set in Table 1A, and each solution was subjectedto rigid body refinement, using first one molecule, thenone helix, per rigid group. Cross-crystal averaging wascarried out among Native-2, EMTS-1 and EMTS-2,together with solvent flattening, 2-fold Ncs averagingand histogram matching, in DMMULTI.138 The atomic

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model was rebuilt using O48 into the averaged map ofNative-2.The model was refined using cns22 with simulated

annealing and group B-factors. Ncs restraints wereapplied between the two molecules in the asymmetricunit, but the restraints were relaxed in the loop regions.This was because in simulated annealing trials, the loopresidues showed systematically larger inter-molecularcoordinate differences than residues in the helices,regardless of whether the NCS restraints have beenimposed over the loops, whereas excluding the loopregions from NCS restraints was found to reduce thefree-R-factor and improve overall stereochemistry.Native-3 was not averaged with the other data sets,

but was subjected to 20 cycles of solvent flattening(46% solvent) and 2-fold Ncs averaging, during whichthe averaging radius was reduced and the resolutionextended from 2.9 A to 2.65 A. The resulting map showedclear densities for the retinal and carbohydrate residues,the palmitoyl modifications to Cys322 and Cys323, as wellas a few tightly bound water and detergent molecules.The 1F88 model used for molecular replacement wasextensively rebuilt, especially the side-chain confor-mations. Coordinates for the retinal and carbohydratemoieties and cytoplasmic loops were taken from thepartially refined Native-2 model and rebuilt. Refinementagainst Native-3 data was done using simulatedannealing and individual B-factors, under Ncs restraintsimposed over the helical segments. When the free-R-factor decreased to 24.5%, ordered water moleculeswere located in the difference map using the water-picktask file in CNS. After another round of refinement withindividual B-factors, NCS copies of the bound watermolecules were located in the sigma-weighted 2FoKFcmap and confirmed in the solvent-flattened EDEN23 map(kindly calculated by Dr Luca Jovine). Detergent andphospholipid molecules were added during refinement.Geometric parameters for the 11-cis-retinal were taken

from the small molecule structure,20 except the 12-s-cisbond was made trans, and parameters for the protonatedSchiff base were taken from the retinylidene structure.21

In early rounds of the refinement, dihedral angles in thepolyene section of retinal were tightly constrained to theplane using energy constants for the tryptophan ring(500 kcal/mol), but for dihedrals about the C6–C7,C11–C12 and C12–C13 bonds the energy constants werereduced tenfold. After addition of water molecules, thetight dihedral restraints along the polyene were relaxed to100 kcal/mol.Initial models for the detergent molecules LDAO and

C8E4, and the hydrocarbon chains of phospholipids weretaken from structures in the Protein Data Bank asprovided by the HIC-UP web site†. Topology parametersfor these groups and for the thioester linkage ofpalmitoylated cysteine were derived from structures inthe Cambridge Small Molecule Database.

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UNCOMolecular replacement from an EM model

From 2D crystals of bovine rhodopsin imaged byelectron cryomicroscopy, a three-dimensional map hasbeen calculated to 5 A resolution in the plane and 13.5 Anormal to it.10 The seven transmembrane helices appearas density rods of unknown azimuthal orientations. Toreduce the distortion caused by the missing cone in datacollected from 2D crystals, this map was subjected to one

† http://alpha2.bmc.uu.se/hicup/


ED PROOF

cycle of solvent-flattening using the program dm139 and asolvent content of 75%, which was estimated from thevolume per molecule in the P31 crystal16 and a unit cellthickness of 200 A in the 2D crystal.The solvent-flattened EM map was re-calculated to 6 A

resolution for skeletonisation.140 The bones within theboundary of a single rhodopsin molecule were edited inO to follow the high density ridges along the helix axes,and they were annotated in O for the helices represented,in accordance with the model of helix arrangementproposed by Baldwin et al.12 Using the bone_mask optionin O and a 6 A radius for the bones atoms, a molecularmaskwas formed. Themaskwas edited usingMama141 toremove internal cavities, and converted to the Ccp4format on a 1 A sampling grid using Mama2ccp4.136

The solvent-flattened EM map was interpolated ontothe 1 A grid inside the molecular mask using Maprot,142

to yield the masked density of a single rhodopsinmolecule. The centre of gravity for the masked regionwas shifted to the origin of a P1 unit cell that has 150 Acell edges and 908 cell angles, by editing the map headerusing the program Imedit in the MRC image processingsuite.143 The asymmetric unit was filled with zerosoutside the mask, using the program Mapmask.136 Thisdensity of a single rhodopsin molecule, cut out fromthe EM map, and placed in a large P1 cell was the“EM model” for molecular replacement.Structure factors for the EM model were calculated

using SFALL.136 Cross-rotation function was calculatedusing Glrf,144 in which the best signal-to-noise ratio wasobtained by using a 28 A radius of Patterson integrationand a resolution limit of 9–5 A. UsingMaprot142 again, theEM model was rotated about its centre of gravity to thecross-rotation angles, and re-interpolated into the actualP31 unit cell at 1 A sampling intervals. Translationsearches were conducted using Tffc145 in both P31 andP32. The molecular orientation found16 was subsequentlyconfirmed by molecular replacement from the 1F88coordinates, once these became available. The molecularorientations found using the EMmodel agreed with thosefound subsequently from the atomic model to within 28.However the map of translation search from the EMmodel showed only streaks rather than discrete peaks.

Docking X-ray coordinates to the EM map

The Ca coordinates of the 1F88 model and of the refinedstructure from this work were first manually aligned withthe “bones” for the EM map, which trace out the helixaxes. Then using the RSR_rigid option in O, in theconvolution mode, the fit of all atoms of the proteinmolecule to the EM density was optimised.

Protein Data Bank accession numbers

Coordinates and structure factors have been releasedthrough the RCSB Protein Data Bank (code 1GZM,1GZM-SF).

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Acknowledgements

We thank the ESRF, SPring-8, APS and DaresburyLaboratory for providing synchrotron radiationfacilities and staff support during data collection,and in particular the ESRF for a long-term award to

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G.F.X. for use of the microfocus beamline ID13. Weare grateful to Drs Richard Henderson, PaulHargrave, Joyce Baldwin, Harry Powell and AlexeiMurzin for helpful discussions; Luca Jovine forcalculating the EDEN map from partially refinedcoordinates; Richard Henderson, Phil Evans,Andrew Leslie and Dan Oprian for comments onthe manuscript; colleagues at the MRC Laboratoryof Molecular Biology for encouragement. Weacknowledge the use of the GPCRDB data base†.

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Documents

Structure of Bovine Rhodopsin in a Trigonal Crystal Form